Exposure to a variety of chemical agents that arise from both endogenous and exogenous sources, as well as exposure to ionizing and ultraviolet radiation, produces crosslinks between DNA and protein molecules, in addition to the more extensively characterized individual DNA or protein adducts. All of these agents are either known or suspected carcinogens and as such, pose significant health risks to exposed human populations. Despite the pervasiveness of these DNA-protein crosslinks (DPCs) and their probable causative role in some cancers, age-related macular degeneration and some neurodegenerative diseases such as Parkinson's, there exists a paucity of data concerning the biological processing of these lesions, because until recently, there have been no mechanisms available to create site-specific, protein-specific lesions in DNA. In order to establish a fundamental understanding of cellular responses to the presence of DPCs, herein for the first time, methodologies are developed that create site-specific DPCs that are located in the major and minor grooves of DNA and along the sugar-phosphate backbone. The availability of DNAs containing site-specific and protein-specific DPCs will enable the testing of hypotheses on how these lesions are replicated and repaired in mammalian cells. Specific Aim (1) lays the foundation for the construction and physical characterization of defined DNAs containing a variety of DPC lesions; strategies are developed to create various sized DPCs (600 - 120,000 Da) in DNAs and characterize their modulation of the DNA structure by analyses of bend angles, footprint, and thermal destabilization. Specific Aim (2) will characterize in vitro replication and repair of DNAs containing these DPC lesions, using both prokaryotic and mammalian NER assays, DNA replication bypass analyses, and DNA helicase unwinding studies. Specific Aim (3) evaluates the mutagenic potential and in vivo repair pathways for DPCs by engineering these lesions into single- and double-stranded vectors and replicating them through repair proficient and deficient cells; mutational spectra will be analyzed for these site-specific lesions.